Signal transduction networks are a crucial part of the circuitry by which a cell regulates its growth and developmental program and its response to its environment. Faulty or malfunctioning signaling pathways underlie the molecular pathology of many diseases, particularly cancer. Protein kinase cases are central components of many signaling pathways. Often, the transmission of different upstream signals involves common protein kinases, yet elicits distinct (and appropriate) outcomes. On the other hand, distinct pathways may contain highly similar protein kinase cascades, yet remain appropriately insulated from one another. How specificity form cellular to signal response is maintained in such cases is not well understood, and a fundamental problem in cell biology. This proposal presents experiments that address the role of a high-affinity protein-protein interactions in the specificity of signal transmission between mitogen-activated protein kinases (MAPKs, also called extracellular-signal regulated kinases, or ERKs) and MAPK/ERK kinases (MEKs). Specifically, a short, evolutionarily conserved motif ( the MAPK docking site) has been identified on MEKs that, in yeast, has been shown to mediate high-affinity binding to the cognate MAPKs. Specific aims are to determine if the docking sites of human MEK1 and Mek2 bind to the cognate human MAPKs, to characterize the amino acid residues on the yeast and human MEKs and MAPKs that mediate this high-affinity binding, and to use this information to construct appropriate mutants to learn more about the in vivo functions of MEK/MAPK docking. In so doing, we will address two (non-exclusive) hypotheses about the role of high-affinity protein-kinase substrate interactions in protein kinase signaling the transmission hypothesis and the specificity hypothesis. Inappropriate MAPK activation, such as that evoked by the well known oncoproteins Ras and Raf, is sufficient for the neoplastic transformation of cultured mammalian cells and consequent tumorigenesis. The research proposed herein should improve our understanding of the signal transmission and signal specificity in MAPK cascades. More generally, this research could lead to better understanding of how protein kinases find their targets, improve our ability to predict targets, and may ultimately suggest novel approaches to therapy based upon the modulation of protein kinase interactions.